Utilization of RESOLV with polymer to produce prazosin hydrochloride nanoparticles and optimization of the process parameters

In this study, rapid expansion of a supercritical solution into a Liquid Solvent (RESOLV) was used for the first time to produce pharmaceutical nanoparticles of Prazosin hydrochloride (PRH). The Taguchi method (robust design) was utilized to design the experiments and ensure obtaining the optimal process conditions. The pressure (15–25 MPa), temperature (308–328 K) and nozzle diameter (300–700 μm) effects on the morphology and size distribution of the resulting particles were also examined. The size of the particles decreased from about 40 μm to the range of (252–418 nm). FTIR, DLS, FESEM, XRD, DSC were used to characterize the primary and processed PRH particles. According to DSC investigations, RESOLV-produced PRH showed lower crystallinity than original PRH.


RESOLV apparatus and procedure
Figure 1 provides the schematic of the RESOLV method device, including units for extraction and precipitation.CO 2 enters the extraction cell, which has an internal volume of 50 ml, to reach the desired maximum pressure (40 MPa).The components of RESOLV device include CO 2 tank, Valve, filter, Air Compressor, Haskel pump (type-CA 91502, Burbank, CA, USA company), cooling and heating systems (refrigerator and oven, respectively), heather, orifice nozzle, participation sampler, flow meter, measuring instrument, and control panel.
First, 2 g of PRH is poured into the cell in a homogeneous form along with glass beads with a diameter of 2 mm to fill it.Then 1mL of ethanol as a cosolvent is added to the cell and the desired temperature (308, 318, and 328 K) and pressure (15, 20, 25 MPa) are adjusted.In the extraction device, two filters are placed at both ends of the cell, and the distance between the nozzle head and the solution is 2 cm.A refrigerator reduces the temperature of the CO 2 to about 263 K at the process onset to prevent CO 2 gas formation in the piston and its locking in the high-pressure pump.After that, CO 2 enters the extraction cell with an internal volume of 50 ml using a high-pressure reciprocating pump to ensure the optimum pressure.A closely controlled oven with a ± 0.1 K temperature set the desired temperatures.After a static time (here set to 60 min), the saturated PRH-CO 2 was transferred through a preheated 1/8" stainless steel tube and micrometer valve to an orifice nozzle to avoid clogging of the nozzle during the expansion phase.In addition, the dynamic time of rapid expansion was 180 min through the nozzle to solution with concentration 3 mg/ml (45 mg of PVP and 15 ml of deionized water).A few drops were then placed on the slide to form the layer.After that, it was placed in the oven to remove water from the composite and PRH samples were prepared for FTIR, DLS, FESEM, XRD and DSC analysis.

Particle characterization
The particles produced from the RESOLV process underwent characterizations by FTIR spectroscopic analysis, XRD, FESEM, DSC, and DLS.DSC analysis highlights the melting point values related to the original and processed drugs.Here, 5 mg of the sample is heated at a 10 °C/min heating rate over a 30-300 °C temperature range of temperature range in a standard aluminum pan under argon atmosphere with a 20 ml/min flow.The X-ray diffractometer of the original and processed patterns was checked utilizing CU-Ka radiation (λ = 0.154 nm under room temperature in 2 different values at a range of 5-80 θ, and this crystal structure, phase purity and average crystal size.FTIR analysis is used for the shape and position evaluation of absorption peaks.Thus, 3 mg PRH is coupled with spectral grade KBr (300 mg) by mortar and pestle, followed by the sample compression to  obtain a KBr disk made on a Hitachi.FESEM analyses was used to investigate particle shape and surface profile.
In FESEM imaging, gold-palladium alloy is employed to coat powder samples.For this purpose, in each run, a few drops of the suspended nanoparticle mixture were placed on a glass slide and placed in an oven to dry.The nanoparticle size distribution was determined by DLS using a He.N 2 laser with a wavelength of 623 nm and a power of 10 mW at a scattering angle of 90° as a light source.Approximately 1 mg of PRH was dissolved in 10 ml of deionized water prior to DLS analysis.Moreover, the loaded drug was calculated based on the method reported by Ameri et al. 38 and reported in Table 3.

Experimental design of process parameters
Design of Experiments (DoE) method was used to determine the optimization values of parameters affecting PRH production.Parameter design contributes essentially to the Taguchi methodology to ensure higher quality with no increase in the costs.This research investigated DoE within RESOLV considering three orthogonal Table 3. Operation conditions of the RESOLV processes and quantitative results.parameters in three levels (L-9).Preliminary tests were considered to choose the key factors and their relevant levels.The size of the nanosized particles was investigated by RESOLV according to the parameters of pressure in the range (15-25 MPa) and temperature (308-328 K) and nozzle diameter of (300, 500, 700 μm).

Results and discussion
PRH solubility in SC-CO 2 under 308-338 K temperature and 120-270 bar pressure ranges was investigated spectroscopically 61 .The obtained results showed the mole fraction between 1.59 × 10 −5 and 7.2 × 10 −5 .RESOLV can be a favorable method for the formation of PRH micro and nanoparticles.Several parameters may influence particle size, particle size uniformity, morphology, and the medicinal compound's dissolution rate when using RESOLV.Temperature, pressure, nozzle diameter, polymer type, nozzle length, solvent content, and flow rate are the main influential parameters.This paper examined the effects of temperature (308-328 K) and pressure (15-25 MPa), nozzle diameter (300, 500, 700 μm), and type of PVP polymer on particle size.The conventional single-factor approach may be very costly and time-consuming at times.From design Taguchi (orthogonal arrangement L-9) it has been used for a better effect of the mentioned variables on the production of nanoparticles.The obtained outputs are presented in Tables 3 and 4 and Figs. 2 and 3.All measurements were repeated three times to obtain a more reliable result (values are reported by particle size uniformity).

Effect of operating conditions on particle size and particle size distribution in RESOLV
The RESOLV method included ANOVA utilizing the experimental results in Table 3 to examine the importance of pressure (15, 20 and 25 MPa), temperature (308, 318, 328 K) and nozzle diameter (300, 500 and 700 μm).Hence, parameters with smaller p-(p < 0.05) and larger F-values had a significant impact on the RESOLV process.Table 4 highlights the statistical significance of pressure (p = 0.0046), temperature (p = 0.0048), and nozzle diameter (p = 0.0133) in terms of reduced particle size.Higher effectiveness of pressure and temperature than nozzle diameter in reducing size is also confirmed by the results.Various statistical indices, including coefficient of determination (R 2 ), adjusted and predicted coefficients of determination (R 2 adj and R 2 pred , respectively), sufficient accuracy, and coefficient of variation (CV), were utilized for statistical significance investigations.Values of R 2 (0.9979), R 2 adj (0.9919), and R 2 pred (0.9592) can highlight the importance of the method and predict the model's adequacy.Pressure, temperature, and nozzle diameter are the most effective parameters explaining 0.9919 of the total size reduction variation, as shown by the adjusted model's R 2 value.The resulting value of sufficient accuracy (34.12, i.e., more than 4) shows an excellent signal-to-noise ratio for movement in the designed space.The low CV value (4.06) confirms the test reliability.ANOVA was utilized to analyze the experimental results obtained for the pressure, temperature, and nozzle diameter impacts on PRH particle size (Table 4) to determine the best particle size-parameters connection.
CV shows the exact degree of comparison of treatments, indicating that the experimental results are appropriately accurate and reliable by CV = 3.9.The estimated R 2 for PRH particle size is 0.9979.As shown in Table 4, the statistical model can justify 99.79% of the response changes.The predicted and adjusted R 2 show reasonable agreement, with respective values of 0.9592 and 0.9919.The large value of determined coefficients (R 2 adj = 0.9919) indicates a very significant model break.The predicted residual error sum of squares (PRESS = 1607.45)presents a measure of model fit for the search points in the design and can be estimated by squaring the difference between the actual predicted values at each point and the sum of squares across the total set of points.Lower PRESS values represent a better model fit into the data points.

The effect of parametric conditions
All the facts presented above support Taguchi as an appropriate model for the present research.Important conditions and parameters are pressure, temperature and nozzle diameter.The design responses for the independent parameters, which include the associations of particle size and parameters, are shown in Fig. 2. Data in Table 3 attribute the highest importance to pressure in reducing the size of particles.When the pressure increases from 15 to 25 MPa, the particle size decreases from 418.2 to 252.4 nm, which is clearly evident in Fig. 2.The detailed data and discussions on the pressure impact on the size of particles were previously provided.Noteworthy, with increasing pressure, the strength of the solvent increases, consequently enhancing the nucleation rate and the production of fine particles.The temperature impact on the RESOLV process was studied as a thermodynamic factor.The graphs in Fig. 2, show the temperature, pressure, and nozzle diameter impacts on the size of particles.www.nature.com/scientificreports/ The first two parameters show an interactive effect on particle size, making the assessment of temperature effects on the process more difficult than pressure.An increase in the temperature leads to a reduction in the SC-CO 2 density, consequently decreasing the solvent power.On the contrary, an increased sublimation vapor pressure can be observed at higher temperatures, increasing the supercritical fluid's solubility.Therefore, increasing temperature alters SC-CO 2 solubility according to the impacts imposed by solvent density and sublimation vapor pressure 18,62 .As highlighted by the obtained data, increased temperature to 318 K results in larger particles, probably due to the double effects on the particle size.At this temperature, the solvent power effect overcomes the sublimation vapor pressure.Overall, temperature's positive effects can be considered as a higher sublimation vapor pressure under greater temperatures.Another parameter that can affect the particle size production of processed PRH is the nozzle diameter.Experimental tests were carried out with different nozzle diameters: 300, 500 and 700 microns.As shown in Fig. 2, the smallest particles were obtained with a 300 μm diameter nozzle.At a temperature of 308 K with a smaller nozzle diameter (Dn = 300 μm), increasing the nucleation rate increases the risk of coagulation of smaller particles and eventually larger particles are formed.However, with the PVP solution, coagulation did not occur.By increasing the nozzle diameter to 700 µm, a slight increase in particle size was observed.Table 3 shows that at a temperature of 328 and 318 K, the smaller the nozzle diameter used in the RESOLV method, the smaller the size of the particles produced.The optimum values of temperature 308 K, pressure 25 MPa and nozzle diameter 300 μm were determined.These values were predicted for 202.5 nm particles.Therefore, the average particle size was found to be 217.4nm, which was close to the predicted value.

PRH characterization
The structures and morphologies of the original PRH and the RESOLV-PVP nanoparticles were determined by FESEM, and the results are shown in Fig. 3.According to the SEM results of the original PRH, the original PRH consists of irregularly shaped crystals (Fig. 4).The FESEM results show the particle size reduction in the processed samples well with the RESOLV method in Figures b and c.PRH and polymer-drug molecules were mixed in the matrix and precipitated together, and Figures d, e and f show that PRH is trapped in the polymer.(Fig. 3).The analysis of the FTIR spectra recorded in Fig. 5 is related to PRH.By studying the FTIR diagram of the obtained sample and comparing it with the diagrams of two substances, drug and PVP, it is possible to identify the peaks in the total sample of the peaks and those in the two samples of PVP and drug, which are displayed with similar colors in the table, confirming the presence of these materials in the sample.Figure 6 shows the DSC analysis results for pure PRH, pure polymer, and the obtained sample.Pure PRH shows a 275°C melting point temperature (T m ), which is exothermic according to the corresponding peak characteristic, indicating the parent PRH's crystalline nature and highlighting good agreement with previous findings 5,63 .
DSC analysis of pure PVP in Fig. 6 shows that the exothermic transition occurs in the range of 55 to 80 °C, which corresponds to the diffusion of moisture at the adsorption temperature.According to the DSC results of the obtained sample in Fig. 6, there is a slight change in the position and intensity of PRH particles' exothermic melting peak to 258.3° Celsius utilizing PVP, possibly associated with the particles' smaller size and amorphous configuration (Pathak et al. 12,46 ).There is also a difference melting point and peak intensity for RESOLV in PVP solution, which confirms the system's reduced size and amorphous nature.
The DLS graphs of PRH nanoparticles under different conditions (runs 1, 3, 4, 5, 6 and 7) are shown in Fig. 4. The DLS and FESEM results are in good agreement.Based on Fig. 3, the thinnest particle size distribution is related to the run at the lowest temperature.The DLS and FESEM results showed that the micro-scale particle size of unprocessed PRH changed to the nano-scale particles after the RESOLV process.The average particle size of the processed PRH (runs 1, 3, 4, 5, 6 and 7) was determined by DLS analysis to be 372.XRD analysis was used (Fig. 7) to evaluate the crystal structure of PRH, indicating the original PRH to be well crystalline with clear peaks at 5-80 of 2θ diffraction angles.As presented in Fig. 7, RESOLV XRD analysis shows that the processed drug has less crystallinity than the original particles, potentially because of their particle size reduction.However, no obvious peak indicating the crystalline nature of the processed samples was seen, www.nature.com/scientificreports/while there was a broad peak indicating the samples' amorphous nature.These results prove the effectiveness of RESOLV in placing PRH in amorphous state.

Optimum conditions
The optimum values of the process parameters were determined to obtain the smallest particle size using the Taguchi method implemented in the Design Expert software.These values were determined to be a temperature of 308 K, a pressure of 25 MPa, and a nozzle diameter of 300 μm.These values were predicted to yield particles    of 202.5 nm in size.The Taguchi method was used to evaluate the accuracy and validity of the optimization method through experiments, and the average particle size was found to be 217.4nm, which was very close to the predicted value.

Conclusion
This study used the RESOLV method for the first time to control the size and also the size distribution of PRH as a water-insoluble drug.The effects of temperature (308-328 K), pressure (15-25 MPa) and nozzle diameter (300, 500, 700 μm) on particle size were investigated.PRH drug nanoparticles were identified using FTIR, XRD, DSC, FESEM analyses.FTIR and XRD analyzes showed that no changes occurred in the chemical structure of PRH.FESEM results show the reduction of particle size by RESOLV method.According to the DSC results of the sample obtained, there is a slight change in the position and intensity of the exothermic melting peak of PRH particles up to 258.3 °C using PVP, which is probably related to the smaller particle size and amorphous configuration.Besides, the pressure, temperature, nozzle diameter impacts on the morphologic profile, particle size distribution were investigated and underwent optimizations by the design method.In the Taguchi method, the lowest and highest values achieved are 248.2 and 420.3 nm.The lowest value calculated by RESOLV is 252.4 nm and the highest value is 418.2 nm.This experiment is in good agreement with Taguchi's design.A particle size of 202.5 nm was predicted by the Taguchi method.The average particle size was 217.4 nm, which was very close to the expected value when the Taguchi method was used to evaluate the accuracy and validity of the optimized point.

Figure 1 .
Figure 1.Experimental apparatus of the supercritical solution rapid expansion into a liquid solvent.

Figure 2 .
Figure 2. The particle size as a function of temperature-pressure and nozzle diameter (Dn).

Table 1 .
Literature review on impregnation in different contexts (RESOLV).

Table 2 .
The solute Prazosin hydrochloride structure and the corresponding physic-chemical profile.

Table 4 .
Taguchi method adequacy and ANOVA analysis for RESOLV.